Investigating dihydroorotate dehydrogenase inhibitor mediated mitochondrial dysfunction in hepatic in vitro models

• Published in: Toxicology in vitro Vol. 72; p. 105096
• Main Authors: Jones, Samantha W., Penman, Sophie L., French, Neil S., Park, B. Kevin, Chadwick, Amy E.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-04-2021; Elsevier Science Ltd
• Subjects: Autoimmune diseases; Biocompatibility; Biotransformation; Cytotoxicity; Dehydrogenase; Dehydrogenases; Dihydroorotate dehydrogenase; DILI; Dysfunction; Electron transport; HepaRG; Hepatotoxicity; HepG2; Immunological diseases; Inhibitors; Leflunomide; Liabilities; Liver; Medical treatment; Metabolism; Mitochondria; Oxidative metabolism; Toxicants; Toxicity; HepG2; Mitochondria; DILI; HepaRG; Dysfunction; Dihydroorotate dehydrogenase
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2021.105096

Inhibition of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzymatic step in de novo pyrimidine synthesis, has broad immunosuppressive effects in vivo and shows promise as a therapeutic target for the treatment of malignancies, viral infections and auto-immune diseases. Whilst there are numerous DHODH inhibitors under development, leflunomide and teriflunomide are the only FDA approved compounds on the market, each of which have been issued with black-box warnings for hepatotoxicity. Mitochondrial dysfunction is a putative mechanism by which teriflunomide and leflunomide elicit their hepatotoxic effects, however it is as yet unclear whether this is shared by other nascent DHODH inhibitors. The present study aimed to evaluate the propensity for DHODH inhibitors to mediate mitochondrial dysfunction in two hepatic in vitro models. Initial comparisons of cytotoxicity and ATP content in HepaRG® cells primed for oxidative metabolism, in tandem with mechanistic evaluations by extracellular flux analysis identified multifactorial toxicity and moderate indications of respiratory chain dysfunction or uncoupling. Further investigations using HepG2 cells, a hepatic line with limited capability for phase I xenobiotic metabolism, identified leflunomide and brequinar as positive mitochondrial toxicants. Taken together, biotransformation of some DHODH inhibitor species may play a role in mediating or masking hepatic mitochondrial liabilities.
•Mitochondrial dysfunction is a putative mechanism by which DHODH inhibitors elicit adverse hepatic outcomes.•Dihydroorotate dehydrogenase inhibitors cause moderate indications of mitochondrial dysfunction in the HepaRG® cell line.•Brequinar and leflunomide screen as positive mitochondrial toxicants in HepG2 cells.•Mitochondrial toxicity in the HepaRG® cell line may be understated due to the expression of functional CYP450 isozymes.